Fermented food products containing probiotic strains, and method for preparing same

ABSTRACT

The use of at least one sulphur-containing amino acid, at a total concentration of about 5 to 75 mg/ml, in particular of about 5 to about 50 mg/l, in particular of about 5 to about 30 mg/l, in particular of about 5 to about 20 mg/l, in free form, for implementing a method for preparing a fermented food product fermented by ferments containing bifidobacteria, the food product has acceptable sensory properties, contains more than about 5×10 7 , in particular more than about 10 8  bifidobacteria per gram of food product fermented for a shelf lifetime of at least 30 days, in particular a shelf lifetime of at least 35 days, and containing no more than 0.5% of yeast extract or of yeast autolysate.

The invention relates to fermented food products containing probioticstrains, and their preparation process.

The bifidobacteria belong to the dominant anaerobic flora in the colon.The main species present in the human colon are Bifidobacteriumadolescentis, Bifidobacterium bifidum, Bifidobacterium longum sspinfantis, Bifidobocterium breve, Bifidobacterium longum.

The bifidobacteria are probiotic bacteria of choice. Bacteria of thegenus Bifidobacterium are used in numerous products currently on themarket and are often added to dairy products already comprising thestandard bacteria in yogurt (Streptococcus thermophilus andLactobacillus bulgaricus).

The consumption of bifidobacteria is recognized as being beneficial inthe process of re-establishing the normal bifidobacteria population inindividuals having undergone antibiotics therapy. This consumption alsoseems to make it possible to reduce constipation, prevent diarrhoea andreduce the symptoms of lactose intolerance.

Probiotics are live bacteria. The use of these live bacteria in themanufacture of food products such as dairy products is tricky inparticular with regard to the problem of survival of these bacteria inthe product.

80% of the products currently on the market which contain bifidobacteriado not satisfy the criteria making it possible to maintain that theysignificantly improve the intestinal transit of the individualsconsuming them. A daily intake of at least 10⁸ to 10⁹ viable cells hasbeen recommended as the minimum dose making it possible to have atherapeutic effect (Silva A. M., Barbosa F. H., Duarte R., Vieira L. Q.,Arantes R. M., Nicoli J. R., Effect of Bifidobacterium longum ingestionon experimental salmonellosis in mice, J. Appl. Microbiol. 97 (2004)29-37). The required dose can be dependent on the probiotic strain used.

In the case of the production of a bioactive food product containingbifidobacteria the problem therefore arises of obtaining a sufficientpopulation of these bacteria in the product and maintaining it duringthe “life” of the product without resorting to technical solutionscapable of altering the organoleptic qualities of the product.

The problem of the numerical size of the population of probiotic strainsin a fermented dairy product is a known problem (see in particular D.Roy, Technological aspects related to the use of bifidobacteria in dairyproducts, Lait 85 (2005) 39-56, INRA, EDP Sciences).

Several reasons for this problem have been suggested, including thereduction in the population during storage, the disturbed growth ofthese bacteria starting from a certain pH or quite simply the poorability of these bifidobacteria to grow, in particular in milk.

It is known that the fructo-oligosaccharides, certain starches, certainsugars, glycerol and certain yeast extracts have significant bifidogeniceffects. On the other hand oxygen is toxic to certain probiotic strains.

The use of cysteine or ascorbate as an oxygen scavenger has thereforebeen described (A review of oxygen toxicity in probiotic yogurts:influence on the survival of probiotic bacteria and protectivetechniques. Talwalkar & Kailasapathy; Comprehensive Reviews in FoodScience and Food Safety, 3 (3) 117-124; 2004), without it however havingbeen demonstrated that the use of these substances makes it possible toobtain and maintain populations of bifidobacteria at the desired levelsduring storage. Moreover, the potentially negative effect of thecysteine on the final properties of a yogurt has been noted.

Generally, the fermented food products having properties of relativemaintenance of the populations of bifidobacteria during the preservationof said products, and which are described in the literature, do notgenerally have acceptable organoleptic properties, due to the fact inparticular that substances such as yeast extract are present in a highconcentration in the products.

The main purpose of the invention is to provide fermented food productshaving acceptable organoleptic properties and containing a highconcentration of bifidobacteria at the end of the fermentation periodand throughout the preservation period of said fermented food products.

The main purpose of the invention is to provide fermented food productscontaining bifidobacteria in a good physiological state and having asignificant survival rate during the period of preservation of saidfermented food products, in particular up to the use-by date of theproducts.

Another purpose of the invention is to provide preparation processeswhich are simple to implement, making it possible to obtain the aboveproducts.

Another purpose of the invention is to promote the growth of thebifidobacteria in relation to the standard symbioses present in yogurts,these symbioses being constituted in a standard fashion by one or morestrains of Streptococcus thermophilus and of Lactobacillus bulgaricus.

The purposes of the invention are achieved thanks to the surprisingfinding made by the inventors that the incorporation ofsulphur-containing amino acids in the starting substance during thepreparation of fermented food products containing bifidobacteria, in asmall enough quantity not to alter the organoleptic properties of theproducts, makes it possible to obtain rapidly, after fermentation of thepopulations, at least 5.10⁷ or even 10⁸ bifidobacteria per gram ofproduct, and increased survival of the bifidobacteria up to the use-bydate of the products, without necessarily modifying the growth of theother bacterial strains.

The invention relates to the use of at least one sulphur-containingamino acid, at a total concentration of approximately 5 to approximately75 mg/l, in particular approximately 5 to approximately 50 mg/l, inparticular approximately 5 to approximately 30 mg/l, in particularapproximately 5 to approximately 20 mg/l in the free form, for theimplementation of a process for the preparation of a fermented foodproduct using ferments containing bifidobacteria, which fermented foodproduct has acceptable organoleptic properties, contains more thanapproximately 5.10⁷, in particular more than approximately 10⁸bifidobacteria per gram of fermented food product for a preservationperiod of at least 30 days, in particular at least 35 days and does notcontain more than 0.5% of yeast extract or yeast autolysate.

By “sulphur-containing amino acid” is meant cysteine (L-cysteine) ormethionine as well as their derivatives, optionally in the form of asalt.

In particular there can be used according to the invention monohydratedL-cysteine hydrochloride (monohydrated (R)-2-amino-mercaptopropionicacid monohydrochloride) or L-methionine((S)-2-amino-4-methylthio-butyric acid), of the respective formulae:

By “in the free form” is meant amino acids which are not bound to otheramino acids by a peptide bond within peptides, polypeptides or proteins.

Preferably, the sulphur-containing amino acids according to theinvention are used in reduced form, i.e. the sulphhydryl group —SH isreduced. This preferred form of the sulphur-containing amino acidstherefore excludes in particular cystine, the oxidized form of cysteineinvolving the combination of two cysteines via a disulphide bridge.

The bifidobacteria being substantially without proteolytic activity, itis advantageous to use the abovementioned amino acids in the free formso that they can be directly assimilated by the bifidobacteria.

The sulphur-containing amino acid or acids used according to theinvention are advantageously filtered beforehand and/or autoclaved (orpasteurized, i.e. treated at a temperature above 50° C.) and/orirradiated, in order to take account of the constraints of use asregards microbiological contamination, i.e. so that they aresubstantially without microbial contaminants.

If the sulphur-containing amino acids are used at a concentration above75 mg/l, a degradation of the organoleptic properties of the foodproduct is noted.

If the sulphur-containing amino acids are used at a concentration below5 mg/l, the population of bifidobacteria greater than 5.10⁷ or 10⁸ CFUper gram of product cannot generally be maintained during thepreservation period of the product.

It should be noted that the concentration of sulphur-containing aminoacids used according to the invention relates to the sulphur-containingamino acid or acids especially added during the preparation of theproducts. This concentration does not take account of the possiblebacterial production of sulphur-containing amino acids during thepreparation nor even of the quantity of sulphur-containing amino acidsin the free form which are naturally present in the starting substancewhich serves to prepare the food product (for example in milk) or in theadjuvants which can be used during the preparation.

The typical concentration of sulphur-containing amino acids present inmilk is 100 to 1300 mg/l including approximately 260 mg/l cysteine and1020 mg/l methionine (Handbook of Milk composition, 1995, AcademicPress). It should be noted that the vast majority of thesesulphur-containing amino acids present in milk is in the bound form inpeptide or protein chains.

By “ferments” is meant a set of bacteria, in particular bacteriaintended for fermentation and/or bacteria with probiotic value.

By “acceptable organoleptic properties” is meant in particular theabsence of an undesirable sulphur-type taste, as determined by astandard sensory analysis test, which can correspond to the protocoldescribed hereafter.

The sensory mechanism starts with the generation of a stimulus followingthe consumption of a product. This stimulus allows a perception which isdependent on genetic and physiological factors in the individualconsumer. This perception is then verbalized (a list of words isproposed to the consumer) then quantified (use of ranges). The consumerthen gives an overall assessment of the product that he has consumed(this assessment is influenced by his culture, his experiences) and sayswhether or not he would be prepared to buy this product (data such ascost, communication about this product can then be provided).

Sensory analysis is a science based on perception (physiological andpsychological) involving the five senses (taste, smell, sight, hearing,touch) and using very rigorous protocols.

The consumers constituting the panel who carry out the sensory analysesare selected for their sensory abilities, their abilities in terms ofverbalization, their abilities in terms of use of ranges for anassessment and their abilities to work in a group (in order to obtain aconsensus). It is absolutely necessary to verify that the assessments ofthe panel members are repeatable, reproducible, with a homogeneity interms of discrimination and in terms of classification. Tests making itpossible to verify these pre-requisites are repeated several times. Thechoice of products is made according to three main criteria: accordingto the age of the product (products of the same age are chosen), theseproducts must be representatives in the case of a standard assessmentand the products are homogeneous (few differences between them). Theseproducts are presented anonymously and in coded form, in a certain orderand homogeneously (same temperature etc.).

The environmental conditions of the sensory analysis are important: theconditioned air, the lighting, the sound environment, the decoration(neutral if possible) and the odour of the room in which the analysis iscarried out should be standardized. The panel members are separated bycubicles. They should not smoke, consume coffee or menthol in the hourspreceding the analysis session. They should also not wear perfume ormake-up.

At the end of this analysis, a product is to be considered as having“acceptable organoleptic properties” if the panel members have notdetected an undesired sulphur-type taste in this product.

The preservation or storage period of the fermented food product is theperiod which immediately follows the end of the process of preparationof the fermented food product and its packaging. During thispreservation period the fermented food product is usually preserved at atemperature comprised between approximately 4 and approximately 10° C.

The abovementioned fermented food product contains more than 5.10⁷, inparticular more than 10⁸ bifidobacteria per gram of fermented foodproduct in particular for a preservation period of at least 40 days.More particularly the above-mentioned fermented food product containsmore than 5.10⁷, in particular more than 10⁸ bifidobacteria per gram offermented food product up to the use-by date of the product.

The use-by dates depend on the legal preservation periods fixed bycurrent legislation, which can typically vary from 15 to 50 days fromthe date of production. By way of example, the legal preservation periodis generally 30 days for fresh dairy products.

A population of bifidobacteria which is greater than or equal to 10⁸CFU/g at the use-by date of product preserved between 4 and 10° C. canbe considered a sufficient population of bifidobacteria given themedical recommendations relating to the provision of bifidobacteria infood.

By “does not contain more than 0.5% of yeast extract or yeastautolysate”, is meant in particular that the abovementioned fermentedfood product does not contain more than 0.5% of yeast extract or yeastautolysate at the end of its preparation process and/or that theabovementioned fermented food product does not contain more than 0.5% ofyeast extract or yeast autolysate for the preservation period of atleast 30 days, in particular at least 35 days, in particular at least 40days or up to the use-by date of the abovementioned fermented foodproduct. Moreover, the abovementioned fermented food product no longercontains a quantity greater than 0.5% of yeast extract or yeastautolysate during the process for the preparation of the product, and inparticular at the time of the inoculation of the bacteria and throughoutthe fermentation.

By “yeast extract” and “yeast autolysate” is meant concentrates ofsoluble compounds of yeast cells. In this regard reference may be madein particular to the article “Yeast extracts: production, properties andcomponents” by Rolf Sommer (9^(th) International Symposium on Yeasts),from which the information below is extracted.

Yeast extracts are mainly produced by autolysis, i.e. cell hydrolysis iscarried out without the addition of other enzymes. The yeast extract oryeast autolysate are used mainly in the fermentation industry and in theagri-food industry. The main raw material used in order to produce theyeast extract is constituted by yeasts with a high concentration ofproteins (strains of Saccharomyces cerevisiae) cultured on media basedon molasses or is constituted by yeasts from debittered beer (strains ofSaccharomyces cerevisiae or Saccharomyces uvarum). Other raw materialsused are yeasts such as Kluyveromyces fragilis (fermented on lactoserum)or Candida utilis (cultured on carbohydrate-rich waste originating fromof the timber industry or on ethanol) or also special strains of baker'syeasts, in order to produce yeast extract containing 5′-nucleotides.

Autolysis is the dissociation process most frequently used in theproduction of yeast extract. During this process, the yeasts aredegraded by their own endogenous enzymes. The autolysis process can beinitiated by an osmotic shock or controlled temperature, causing celldeath without inactivating the endogenous enzymes (in particular theproteases). A controlled pH, the temperature and the duration of theautolysis are decisive factors in a standardized autolysis process. Byadding salts or enzymes (for example proteases or mixtures of proteasesand peptidases) relative to the “standard” autolysis, the proteindegradation of the yeast cells can be controlled.

Besides autolysis, the yeast extract can be produced by thermolysis (forexample by boiling the yeasts in water at 100° C.), plasmolysis(treatment with strong saline solutions at a temperature below 100° C.)and mechanical degradation (high-pressure homogenization or grinding).

Then the soluble compounds are separated from the insoluble cell wallsand concentrated with an evaporator with stirring or falling filmevaporator, followed by optional stages of filtration, concentrationunder partial vacuum and rapid sterilization. Three types of yeastextract exist: liquid yeast extract (dried matter: 50 to 65%); viscouspaste-type yeast extract (dried matter: 70 to 80%); dry yeast extractpowder.

Taking the example of a standard yeast extract powder used in thefermentation industry, the composition is the following:

Protein content: 73-75% Sodium: less than 0.5% Polysaccharides: lessthan 5% Oligosacharides: less than 1% Lipids: less than 0.5%

The protein content is typically distributed as follows:

Free amino acids: 35-40% Di, tri and tetrapeptides (MW < 600 Da): 10-15%Oligopeptides (MW of 2000-3000 Da): 40-45% Oligopeptides (MW of3000-100000 Da): 2-5%

The typical cysteine content is 0.45%, and the typical methioninecontent is 1.12% (1.08% in the free form).

The invention relates to the use of at least one sulphur-containingamino acid, at a total concentration of approximately 5 to approximately30 mg/l, in particular approximately 10 to approximately 15 mg/l, inparticular approximately 12 to approximately 15 mg/l, and in particular12.5 mg/l, in the free form, for the implementation of a process for thepreparation of a fermented food product using ferments containingbifidobacteria, which fermented food product has acceptable organolepticproperties, contains more than approximately 5.10⁷, in particular morethan approximately 10⁸ bifidobacteria per gram of fermented food productfor a preservation period of at least 30 days, in particular at least 35days and does not contain more than 0.5% yeast extract or yeastautolysate.

Moreover, the invention also relates to a fermented food product, havingacceptable organoleptic properties, containing ferments comprising morethan approximately 5,107, in particular more than approximately 10⁸bifidobacteria per gram of fermented food product for a preservationperiod of at least 30 days, in particular at least 35 days and having atotal concentration of sulphur-containing amino acids in the free formof approximately 5 to approximately 50 mg/l, in particular approximately5 to approximately 30 mg/l, in particular approximately 5 toapproximately 20 mg/l, in particular approximately 10 to approximately15 mg/l, in particular approximately 12 to approximately 15 mg/l, and inparticular 12.5 mg/l.

More particularly, said fermented food product contains fermentscomprising more than approximately 5.10⁷, in particular more thanapproximately 10⁸ bifidobacteria per gram of fermented food product fora preservation period of at least 40 days or up to the use-by date ofthe fermented food product.

Advantageously, the fermented food product as defined above is such thatthe ratio of the number of bifidobacteria contained in the fermentedfood product at the end of the preservation period to the number ofbifidobacteria contained in the fermented food product at the start ofthe preservation period of at least 30 days, in particular at least 35days, is approximately 0.2 to approximately 0.8, in particularapproximately 0.3 to approximately 0.7, in particular approximately 0.4to approximately 0.5.

In other words the survival rate of the bifidobacteria contained in thefermented food product between the start of the preservation period(i.e. the end of the preparation process) and the end of thepreservation period is comprised between 20 and 80%, in particularbetween 30 and 70%, and in particular between 40 and 50%.

Said preservation period is at least 30 days, in particular at least 35days, but more particularly at least 40 days or extends at least up tothe use-by date of the fermented food product.

The invention also relates to a fermented food product preserved for apreservation period of at least 30 days, in particular at least 35 days,at a temperature of approximately 4 to approximately 10° C., havingacceptable organoleptic properties and containing ferments comprisingmore than approximately 5.10⁷, in particular more than approximately 10⁸bifidobacteria per gram of fermented food product.

More particularly the invention relates to a fermented food productpreserved for a preservation period of at least 30 days, in particularat least 35 days, in particular at least 40 days, at a temperature ofless than 12° C. or less than 10° C., having acceptable organolepticproperties and containing ferments comprising more than approximately5.10⁷, in particular more than approximately 10⁸ bifidobacteria per gramof fermented food product.

Preferably, the invention relates to a fermented food product as definedabove containing approximately 5 to approximately 50 mg/l, in particularapproximately 5 to approximately 30 mg/l, in particular approximately 5to approximately 20 mg/l, in particular approximately 10 toapproximately 15 mg/l, in particular approximately 12 to approximately15 mg/l, and in particular 12.5 mg/l, of sulphur-containing amino acidsand in particular approximately 5 to approximately 50 mg/l, inparticular approximately 5 to approximately 30 mg/l, in particularapproximately 5 to approximately 20 mg/l, in particular approximately 10to approximately 15 mg/l, in particular approximately 12 toapproximately 15 mg/l, and in particular 12.5 mg/l of cysteine and/orapproximately 5 to approximately 30 mg/l, in particular approximately 5to approximately 15 mg/l, of methionine.

In order to determine the cysteine, it is possible to use an amino acidanalyzer such as the L-8800 High Speed Amino Acid Analyzer (Hitachi HighTechnologies). This analyzer combines ion-exchange chromatography withcalorimetric detection at two wavelengths (570 and 440 nm) afterreaction with ninhydrin. It is also possible to use gas chromatographycoupled with mass spectrometry or high-performance liquid chromatographycoupled with fluorimetric detection.

The more particular use of cysteine is advantageous as it gives riseexperimentally to a better bifidogenic effect than methionine.

The more particular use of methionine is advantageous as its cost islower than that of the use of the cysteine.

Advantageously, said fermented food product contains less thanapproximately 0.5% (w/w) of substances containing more thanapproximately 1.7% of sulphur-containing free amino acids.

More particularly said fermented food product contains less thanapproximately 0.5% (w/w) of yeast extract and/or yeast autolysate and/ormilk, plant or soya protein hydrolysate.

The possible presence of yeast extract or yeast autolysate typesubstances is easily detectable in the product by known methods. Inparticular, the glucans or the mannans provided by these substances aredetectable. For example, the glucans and mannans being fibres, it ispossible to use the total dietary fibre determination method,recommended by the AFSSA [French Agency for Food Safety] (method AOAC985.29). The addition of yeast extract or a similar substance must alsoresult in a complete modification of the content of all of the 20 aminoacids in the product, as well as in a modification of the concentrationof vitamins and minerals, relative to the normal composition of theproduct (for the example of milk, reference may in particular be made tothe Handbook of milk composition, 1995, Academic Press).

According to a preferred embodiment, the bifidobacteria contained in thefermented food product as defined above are of the type Bifidobacteriumanimalis, in particular Bifidobacterium animalis animalis and/orBidifobacterium animalis lactis, and/or Bifidobacterium breve and/orBifidobacterium longum and/or Bifidobacterium infantis and/orBifidobacterium bifidum.

Advantageously, the fermented food product as defined above is based onplant juice and in particular fruit juice or vegetable juice such assoya juice, or on a dairy product, and in particular on cow's milkand/or on goat's milk.

Said fermented food product can also be based on sheep's milk, camel'smilk or mare's milk.

By plant juice is meant a juice produced from plant extracts, inparticular soya, tonyu, oat, wheat, maize etc.

Examples of vegetable juice are: tomato juice, beet juice, carrot juiceetc.

Examples of fruit juice are: apple, orange, strawberry, peach, apricot,plum, raspberry, blackberry, gooseberry, pineapple, lemon, citrus fruit,grapefruit, banana, kiwi fruit, pear, cherry, passion fruit, mango,exotic fruit juice, multifruit juice etc.

According to a preferred embodiment, the ferments of the fermented foodproduct as defined above contain lactic bacteria, in particular one ormore bacteria of the genus Lactobacillus spp. and in particularLactobacillus delbrueckii bulgaricus and/or Lactobacillus casei and/orLactohacillus reuteri and/or Lactobacillus acidophilus and/orLactobacillus helveticus and/or Lactobacillus plantarum, and/or bacteriaof the type Lactococcus cremoris and/or Streptococcus thermophilusand/or Lactococcus lactis and/or one or more bacteria of the genusLeuconostoc.

Advantageously, the fermented food product as defined above is such thatthe proportion of bifidobacteria in the ferments is approximately 20 toapproximately 80%, in particular approximately 30 to approximately 70%,in particular approximately 40 to approximately 60%, and in particularapproximately 50%.

By “proportion of bifidobacteria in the ferments” is meant theproportion of bifidobacteria relative to the total number of bacteriaincluded in the fermented food product, i.e. relative to all of thebifidobacteria and other bacteria, in particular the bacteriaLactococcus, Lactobacillus, Streptococcus etc.

The good numerical balance between the bifidobacteria and the otherbacterial strains in the fermented food product at the end of thepreparation process, and the substantial maintenance of this balancethroughout the preservation period, are essential guarantees of thequality of the food product.

A proportion of 50% bifidobacteria constitutes a good compromise betweenthe problem of cost (the bifidobacteria are expensive) and the problemof obtaining a correct population of bifidobacteria.

According to a preferred embodiment, the fermented food product asdefined above is presented in the form of a stirred fermented foodproduct or a fermented food product for drinking or a firm fermentedfood product or an infant fermented food product.

By “stirred [ . . . ] product” is meant a product, in particular a milk,seeded, fermented, mechanically stirred then packaged. The fermentationof such a product is carried out not in a pot but in bulk, in tanks. Thecurd is stirred then cooled down before being packed in pots, which arestored under refrigeration. By curd is meant a coagulate of proteins inparticular of milk.

By “[ . . . ] product for drinking” is meant a product in substantiallyliquid form. A product for drinking is a product which is such that,after the mechanical stirring stage, the product is beaten in the tanksbefore being packaged.

By “firm [ . . . ] product” is meant a product (in particular a milk)seeded and directly packaged in pots where it ferments. After theseeding, the product is packaged in pots. These pots are generallyplaced in an oven for 3 hours. The bacteria reproduce and consume thelactose which is then partially converted to lactic acid which modifiesthe structure of the proteins, forming what is known as a “lactic gel”.Then, the products are placed in a ventilated cooler or cooling tunneland stored at approximately 2-4° C.

By “infant [ . . . ] product” is meant a product suited to an infant'sneeds, with a low protein and fat content.

Said fermented food product can in particular be a yogurt or a firm,stirred or drinking yogurt or a bar containing a dairy substance, kefir,a biscuit with a dairy filling, a water containing probiotics etc

Moreover the invention also relates to a process for the preparation ofa fermented food product from a starting substance, comprising

-   -   a stage of seeding a starting substance, optionally pasteurized,        by inoculation with seeding ferments containing bifidobacteria,        in order to obtain a seeded substance,    -   a stage of fermentation of the seeded substance obtained in the        preceding stage in order to obtain a fermented substance,    -   a stage of incorporation of at least one sulphur-containing        amino acid in the free form at a concentration of approximately        5 to approximately 75 mg/l in particular approximately 5 to        approximately 50 mg/l, in particular approximately 5 to        approximately 30 mg/l, in particular approximately 5 to        approximately 20 mg/l, in particular approximately 10 to        approximately 15 mg/l, in particular approximately 12 to        approximately 15 mg/l, and in particular 12.5 mg/l, this stage        of incorporation being able to occur        -   either before the seeding stage,        -   or substantially simultaneously with the seeding stage,        -   or after the seeding stage and before the fermentation            stage,

providing that the fermented food product does not contain more than0.5% (w/w) of yeast extract and/or yeast autolysate.

By “fermentation” is meant a biochemical reaction which involvesreleasing energy from an organic substrate, under the action ofmicro-organisms. It is a conversion process of a raw material by themicro-organisms, this conversion then producing biomass and metabolites.In particular, lactic fermentation is an anaerobic process of theconsumption of lactose by the bacteria in the ferments, which causes theformation of lactic acid and a lowering of the pH.

The invention follows from the surprising finding made by the inventorsthat the incorporation of sulphur-containing amino acids within theabovementioned ranges, in the absence of yeast extract and/or yeastautolysate or in the presence of a low concentration of the latter,makes it possible to improve the resistance of the bifidobacteria andtheir ability to survive. The bidifobacteria contained in the fermentedfood product at the end of the preparation process of the invention arein a better physiological state than if the stage of incorporation ofsulphur-containing amino acids were omitted, which allows a largernumber of these bifidobacteria to survive during the preservation of thefermented food product which follows.

Cysteine and/or methionine therefore have a specific bifidogenic effect.On the other hand the use of yeast extract and/or yeast autolysate, inparticular at concentrations greater than 0.5% (w/w), has a tendency tostimulate all of the bacteria contained in the fermented food product,which can lead to an imbalance in the bacterial symbiosis to thedetriment of the bifidobacteria, and in favour in particular, if theyare present, of the lactic bacteria. The consequences of this imbalanceare a modification of the pH, a production of acetic acid and/or ofH₂O₂, all events which are detrimental to the quality of the product.

Moreover it should be noted that from a concentration ofsulphur-containing amino acids greater than 30 mg/l, in particular froma concentration of sulphur-containing amino acids greater than 50 mg/l,and more particularly from a concentration of sulphur-containing aminoacids greater than 75 mg/l, a clear degradation of the organolepticproperties of the food products is noted. This degradation is noted bymeans of a standard taste test as described above, which reveals theexistence of a sulphur taste capable of making the products unsuitablefor consumption and marketing. It should be noted that the disagreeablesulphur taste occurs in particular in the case of incorporation ofcysteine and/or of methionine at more than 75 mg/l, or even in certaincases at more than 50 or 30 mg/l, but also when the concentrations ofsulphur-containing amino acids exceed such values due to the presence ofadditional substances, for example yeast extract or yeast autolysate, inparticular at a level of more than 0.5% (w/w).

Another important characteristic of the process of the invention is thatthe incorporation of the ferments containing the bifidobacteria is donedirectly into the starting substance intended to become the fermentedfood product, without necessarily resorting to artificial/syntheticintermediate growth media.

According to a particular embodiment, the process as defined above doesnot comprise a stage of addition of additional substances containing oneor more sulphur-containing amino acids.

According to another particular embodiment, the process as defined abovecomprises a stage of addition of additional substances containing one ormore sulphur-containing amino acids in the free form, the concentrationof sulphur-containing amino acids in the free form in the additionalsubstances being less than approximately 1.7%, preferably less thanapproximately 0.5%, and the concentration of said additional substancesin the fermented food product being less than approximately 0.5%.

More particularly, said stage of addition of additional substances caninvolve addition of a yeast extract and/or yeast autolysate and/or milk,plant or soya protein hydrolysate at a concentration of less thanapproximately 0.5% (w/w).

Preferably, this stage of addition of additional substances takes placebefore the fermentation stage, for example substantially simultaneouslywith the seeding stage and/or simultaneously with the stage ofincorporation of at least one sulphur-containing amino acid.

The benefit of an addition substantially simultaneously with the seedingstage and/or simultaneously with the stage of incorporation of at leastone sulphur-containing amino acid is of a practical nature. In thiscase, the additional yeast extract type substances are at leastpartially degraded during fermentation, as they serve to supplynutrients to the ferments. Thus the concentration of the additionalyeast extract type substances varies during the fermentation.

Advantageously, the process for the preparation of a fermented foodproduct as defined above also comprises a pasteurization stage takingplace before the seeding stage, making it possible to obtain apasteurized starting substance from the starting substance.

By “pasteurization” is meant the method usual in the field of foodpreservation involving a rapid heating without boiling, followed byrapid cooling, making it possible to destroy most of the bacteria whilepartially preserving the proteins.

According to a particular embodiment, the stage of incorporation of atleast one sulphur-containing amino acid takes place before thepasteurization stage, the sulphur-containing amino acid or acids beingincorporated at a concentration of approximately 5 to approximately 75mg/l, in particular approximately 5 to approximately 30 mg/l, inparticular approximately 10 to approximately 15 mg/l, in particularapproximately 12 to approximately 15 mg/l, and in particular 12.5 mg/l.

The benefit of incorporation before the pasteurization stage is of apractical nature.

According to another particular embodiment, the stage of incorporationof at least one sulphur-containing amino acid takes place substantiallysimultaneously with the seeding stage, the sulphur-containing amino acidor acids being incorporated at a concentration of approximately 5 toapproximately 50 mg/l, in particular approximately 5 to approximately 30mg/l, in particular approximately 5 to approximately 20 mg/l, inparticular approximately 10 to approximately 15 mg/l, in particularapproximately 12 to approximately 15 mg/l, and in particular 12.5 mg/l.

The benefit of incorporation substantially simultaneously with theseeding stage is of an economic nature (the sulphur-containing aminoacid or acids are not partially destroyed by any heat treatment orpasteurization before the seeding) and of a practical nature.

According to another particular embodiment, the stage of incorporationof at least one sulphur-containing amino acid takes place after theseeding stage and before the fermentation stage, the sulphur-containingamino acid or acids being incorporated at a concentration ofapproximately 5 to approximately 50 mg/l, in particular approximately 5to approximately 30 mg/l, in particular approximately 5 to approximately20 mg/l, in particular approximately 10 to approximately 15 mg/l, inparticular approximately 12 to approximately 15 mg/l, and in particular12.5 mg/l.

The benefit of incorporation after the seeding stage and before thefermentation stage is of a practical nature and ensures an increasedsurvival of the bifidobacteria during the storage of the product.

It should be noted that in the case where the incorporation of thesulphur-containing amino acid or acids takes place before thepasteurization stage, the quantity of sulphur-containing amino acids tobe incorporated must be increased by approximately 30 to 50% withrespect to the case where this incorporation takes place after theoptional pasteurization stage, i.e. in particular substantiallysimultaneously with the seeding stage or after the seeding stage. Infact, in the first case some of the sulphur-containing amino acids aredestroyed during the pasteurization.

In other words, the top part of the concentration range forsulphur-containing amino acids of 50-75 mg/l which is included withinthe concentration range for sulphur-containing amino acids provided inthe invention relates more specifically to the case where theincorporation of the sulphur-containing amino acids takes place prior toa pasteurization stage.

It should be noted that it is possible to envisage dividing the stage ofincorporation of sulphur-containing amino acids into two or moresub-stages, which can optionally occur at different times in the processaccording to the invention. The concentration of sulphur-containingamino acids which is indicated above then corresponds to the totalconcentration of sulphur-containing amino acids at the end of thedifferent sub-stages of incorporation of sulphur-containing amino acids.

According to a preferred embodiment, the process for the preparation ofa fermented food product as defined above comprises a stage of additionof an intermediate preparation simultaneously with the seeding stage orbetween the seeding stage and the fermentation stage, so as to obtain,from the seeded substance, a completed seeded substance, or after thefermentation stage, so as to obtain, from the fermented substance, acompleted fermented substance, said intermediate preparation comprisinga preparation of fruits and/or cereals and/or additives such asflavourings and colourings, and said stage of addition of anintermediate preparation can take place simultaneously with the stage ofincorporation of at least one sulphur-containing amino acid.

The intermediate preparation can in particular contain thickeners(soluble and insoluble fibres, alginates, carragheenans, xanthan gum,pectin, starch, in particular gelatinized, gelan gum, cellulose and itsderivatives, guar and carob gum, inulin) or sweeteners (aspartame,acesulphame K, saccharine, sucralose, cyclamate) or preservatives.

Examples of flavourings are: apple, orange, strawberry, kiwi fruit,cocoa flavouring etc.

Examples of colourings are: beta-carotene, carmine, cochineal red.

Moreover, the preparation of the abovementioned fruits can comprisefruits which are whole or in pieces or in jelly or in jam, making itpossible for example to obtain fruit yogurts.

The intermediate preparation can also contain plant extracts (soya, riceetc.).

According to another embodiment of the invention, the seeding stagecomprises inoculation with seeding ferments containing approximately 10⁶to approximately 2.10⁸, more particularly approximately 10⁶ toapproximately 10⁷ bifidobacteria, per ml (or per gram) of startingsubstance.

If a quantity of bifidobacteria greater than this range is inoculated,undesired acetic acid type tastes can develop. If a quantity ofbifidobacteria less than this range is inoculated, the final quantity ofbifidobacteria is insufficient.

Advantageously, in the process for the preparation of a fermented foodproduct according to the invention, the bifidobacteria are chosen frombacteria of the type Bifidobacterium animalis, in particularBifidobacterium animalis animalis and/or Bifidobacterium animalislactis, and/or Bifidobacterium breve and/or Bifidobacterium longumand/or Bifidobacterium infantis and/or Bifidobacterium bifidum.

Particularly preferably, in the process for the preparation of afermented food product according to the invention, the bifidobacteriaare chosen from bacteria of the type Bifidobacterium animalis.

Advantageously, in the process for the preparation of a fermented foodproduct according to the invention, the seeding ferments contain lacticbacteria, in particular one or more bacteria of the genus Lactobacillusspp. and in particular Lactobacillus delbrueckii bulgaricus and/orLactobacillus casei and/or Lactohacillus reuteri and/or Lactobacillusacidophilus and/or Lactobacillus helveticus and/or Lactobacillusplantarum, and/or bacteria of the type Lactococcus cremoris and/orStreptococcus thermophilus and/or Lactococcus lactis and/or one or morebacteria of the genus Leuconostoc.

According to an advantageous embodiment of the process for thepreparation of a fermented food product as defined above, the proportionof bifidobacteria in the seeding ferments is approximately 20 toapproximately 75%, in particular approximately 30 to approximately 50%,in particular approximately 35 to approximately 40%, in particularapproximately 37.5%.

By “proportion of the bifidobacteria in the seeding ferments”, is meantthe proportion of the bifidobacteria relative to all of the inoculatedbacteria in total during the seeding stage.

This proportion corresponds to an optimum in terms of cost and finalconcentration of bifidobacteria, given that the higher the concentrationof bifidobacteria at the start, the more competitive they are in termsof growth relative to the other strains in the ferments, and the morerapidly the optimum concentration of bifidobacteria is reached.

According to a preferred embodiment of the process for the preparationof a fermented food product as defined above, the starting substance isbased on plant juice and in particular fruit juice or vegetable juicesuch as soya juice, or on a dairy product, and in particular cow's milkand/or goat's milk.

The starting substance can also comprise sheep's and/or camel's and/ormare's milk.

In the case where the fermented food product is a dairy product, thestarting substance can comprise milk, milk powder, sugar, a mixture ofmilk and plant juice, a mixture of milk and fruit juice, a mixture ofmilk and starch.

Advantageously, the process for the preparation of a fermented foodproduct according to the invention is such that the pasteurized startingsubstance is a pasteurized starting substance, which is held, optionallyhomogenized, and cooled down, obtained from a raw material, said processcomprising, before the seeding stage, the following successive stages:

-   -   a stage of standardization of fatty substances of the raw        material so as to obtain a standardized substance,    -   a stage of enrichment with dried matter of the standardized        substance obtained in the preceding stage, so as to obtain an        enriched substance,    -   a stage of preheating of the enriched substance obtained in the        preceding stage, so as to obtain a starting substance,    -   a stage of pasteurization and holding of the starting substance        obtained in the preceding stage, so as to obtain a pasteurized        and held substance,    -   an optional stage of homogenization of the pasteurized and held        substance obtained in the preceding stage, so as to obtain a        pasteurized, held and optionally homogenized substance,    -   a stage of initial cooling of the pasteurized, held and        optionally homogenized substance obtained in the preceding        stage, so as to obtain a pasteurized starting substance, held,        optionally homogenized, and cooled down.

By “standardization of fatty substances” is meant a stage of bringingthe quantity of fats present in the starting substance to apre-determined level. Enrichment with dried matter involves the additionof proteins and fatty substance in order to modify the firmness of thecurd.

“Holding” involves a rapid thermization of the milk and makes itpossible to destroy the vegetative microbial flora, including pathogenicforms. Its typical duration is from 4 to 10 minutes, in particular from5 to 8 minutes, and in particular approximately 6 minutes.

By “homogenization” is meant the dispersion of the fatty substances inthe milk-type substance into small fat globules. The homogenization iscarried out for example at a pressure of 100 to 280 bars, in particular100 to 250 bars, in particular 100 to 200 bars, in particularapproximately 200 bars. This homogenization stage is purely optional. Itis in particular absent from the production process of products with 0%fatty substances.

According to a particular embodiment, the process for the preparation ofa fermented food product as defined above comprises a packaging stagebetween the seeding stage and the fermentation stage, said packagingstage making it possible to obtain, from the seeded substance obtainedin the seeding stage, a seeded and packaged substance.

This particular embodiment corresponds to the case of the firm-typefermented food products.

More particularly, the process for the preparation of a fermented foodproduct as defined above comprises:

-   -   a stage of seeding a starting substance, optionally pasteurized,        by inoculation with seeding ferments containing approximately        10⁶ to approximately 2.10⁸ bifidobacteria, more particularly        approximately 10⁶ to approximately 10⁷ bifidobacteria per ml of        starting substance, in order to obtain a seeded substance,    -   a stage of packaging the seeded substance obtained in the        preceding stage, in order to obtain a packaged seeded substance,    -   a stage of fermentation of the packaged seeded substance        obtained in the preceding stage, such that the temperature at        the start of fermentation is approximately 36 to approximately        43° C., in particular approximately 37 to approximately 40° C.,        the temperature at the end of fermentation is approximately 37        to approximately 44° C., in particular approximately 38 to        approximately 41° C., and the fermentation time is approximately        6 to approximately 11 hours, in order to obtain a fermented        substance,    -   a stage of final cooling of the fermented substance obtained in        the preceding stage, such that the temperature at the start of        the final cooling is less than approximately 22° C. and the        temperature at the end of the final cooling is approximately 4        to approximately 10° C., so as to obtain a fermented food        product.

According to an alternative embodiment, not involving the preparation offirm-type products, the process for the preparation of a fermented foodproduct according to the invention comprises the following successivestages after the fermentation stage:

-   -   a stage of intermediate cooling of the fermented substance        obtained in the fermentation stage, so as to obtain a pre-cooled        substance,    -   a stage of storage of the pre-cooled substance obtained in the        preceding stage, so as to obtain a stored substance,    -   a stage of final cooling of the stored substance obtained in the        preceding stage, so as to obtain a fermented food product.

According to a preferred embodiment, said fermentation stage is suchthat the temperature at the start of fermentation is of approximately 36to approximately 43° C. and in particular approximately 37 toapproximately 40° C., the temperature at the end of fermentation isapproximately 37 to approximately 44° C. and in particular approximately38 to approximately 41° C., and the fermentation time is approximately 6to approximately 11 hours.

Advantageously, said intermediate cooling stage is such that theintermediate cooling time is approximately 1 hour to approximately 4hours and in particular approximately 1 hour 30 minutes to approximately2 hours and the intermediate cooling temperature is approximately 4 toapproximately 22° C.

Preferably, said storage stage is such that the storage time is lessthan or equal to approximately 40 hours.

Advantageously, said final cooling stage is such that the temperature atthe start of final cooling is less than approximately 22° C. and thetemperature at the end of final cooling is approximately 4 toapproximately 10° C.

According to a preferred embodiment, the process for the preparation ofa fermented food product according to the invention comprises:

-   -   a stage of seeding a starting substance, optionally pasteurized,        by inoculation with seeding ferments containing approximately        10⁶ to approximately 2.10⁸, more particularly approximately 10⁶        to approximately 10⁷ bifidobacteria per ml (or per gram) of        starting substance in order to obtain a seeded substance,    -   a stage of fermentation of the seeded substance obtained in the        preceding stage, such that the temperature at the start of        fermentation is approximately 36 to approximately 43° C., in        particular approximately 37 to approximately 40° C., the        temperature at the end of fermentation is approximately 37 to        approximately 44° C., in particular approximately 38 to        approximately 41° C., and the fermentation time is approximately        6 to approximately 11 hours, in order to obtain a fermented        substance,    -   a stage of intermediate cooling of the fermented substance        obtained in the preceding stage, such that the intermediate        cooling time is approximately 1 hour to approximately 4 hours,        in particular approximately 1 hour 30 minutes to approximately 2        hours and the intermediate cooling temperature is approximately        4 to approximately 22° C., so as to obtain a pre-cooled        substance,    -   a stage of storage of the pre-cooled substance obtained in the        preceding stage, such that the storage time is less than or        equal to approximately 40 hours, so as to obtain a stored        substance,    -   a stage of final cooling of the stored substance obtained in the        preceding stage, such that the temperature at the start of final        cooling is less than approximately 22° C. and the temperature at        the end of final cooling is approximately 4 to approximately        10° C. so as to obtain a fermented food product.

According to a particular embodiment of the process for the preparationof a fermented food product as defined above, an additional stirringstage is provided between the fermentation stage and the intermediatecooling stage, making it possible to obtain, from the fermentedsubstance obtained in the fermentation stage, a stirred fermentedsubstance.

By “stirring” is meant a process of mechanical stirring using a turbineor helical stirrer. It is a stage which determines the oiliness of theproduct in particular the dairy product. If the stirring is too violent,incorporation of air and separation of the serum can occur. If thestirring is insufficient, the product risks subsequently becoming toothick.

According to a particular embodiment, the process for the preparation ofa fermented food product according to the invention comprises, after thefinal cooling stage, a stage of preservation of the fermented foodproduct at a temperature comprised between approximately 4 andapproximately 10° C.

The invention also relates to a fermented food product as obtained fromthe process as defined above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents a comparison of the effects of cysteine and vitamin Con the acidification of milk by the ferment of Example 1. The time inminutes is shown along the x-axis, the pH along the y-axis. Curve A:control without vitamin C or cysteine; curve B: vitamin C; curve C:cysteine.

FIG. 2 represents the development of the population of bifidobacteria inthe control model during preservation at 10° C. Along the x-axis, thepreservation time in days; along the y-axis, the population ofbifidobacteria in CFU/ml. ▪: with 15 mg/l of filtered cysteine; ▴:without cysteine.

FIG. 3 represents the development of the population of bifidobacteria inthe milk as a function of the treatment of the stimulant. X-axis:preservation time in days; y-axis: population in CFU/ml. Conditions: ▪,control without cysteine or methionine; ∘, autoclaved cysteine; ,filtered cysteine; □, autoclaved methionine; dotted curve, filteredmethionine.

FIG. 4 represents the monitoring of the population of bifidobacteria inthe control model during preservation at 10° C. X-axis: preservationtime in days; y-axis: population in CFU/ml. ▪: cysteine at 12 mg/lincorporated before pasteurization; ▴: control without cysteine.

EXAMPLES Example 1 Study of the Mode of Action of Cysteine as aStimulant

A ferment comprising Streptococcus thermophilus (CNCM:I-1630)+Lactobacillus delbreckii ssp. bulgaricus (CNCM:I-1632)+Lactobacillus delbrueckii ssp. bulgaricus (CNCM:I-1519)+Bifidobacterium animalis ssp lactis (CNCM: I-2494) is used.

This example involves studying the mode of action of cysteine as astimulant, and determining whether it has a metabolic or antioxidanteffect.

The growth of bifidobacteria in milk is measured in the presence of asolution of vitamin C (0.5 g/l) completely reducing the oxygen in themedium, and a solution of cysteine (50 mg/l).

Constitution of the product model:

Milex skimmed milk powder supplied by Arla food: 120 g

Water: quantity sufficient for 1 kg

A heat treatment is carried out involving pasteurization for 30 minutesat 95° C. in a bubbling water bath.

The cysteine is supplied by Sigma. The solution is prepared at 500 g/l,filtered on a 0.2 μm Nalgène filter unit. This solution is used injectedsterilely into the pasteurized model for a final concentration of 50mg/l.

The vitamin C is supplied by Sigma. The solution is prepared at 100 g/l,filtered on a 0.2 μm Nalgène filter unit (cat. 156-4020, Nalge EuropeLtd, Belgium). This solution is used injected sterilely into thepasteurized model for a final concentration of 0.5 g/l.

The seeding doses of the product model are given in Table 1 whichfollows.

TABLE 1 Seeding doses Volume for 1 l in μl Control Filtered cysteineVitamin C I-1630 100 100 100 I-1519 + I-1632 220 220 220 I-2494 190 190190 Cysteine 100 Vitamin C 5 ml

The seeding is 5.10⁶ CFU/ml of Streptococcus thermophilus and 5.10⁶CFU/ml of Lactobacillus bulgaricus.

The monitoring of the acidification of the model at 37° C. isrepresented in Table 2 below as well as in FIG. 1.

TABLE 2 Monitoring of the acidification of the model Vitamin C Filteredcysteine Control Ta 86 90 83 Vmax −0.0079 −0.01046 −0.00791 pHm 5.965.82 6.06 Tmax 184 208 180 pH 0 6.5 6.6 6.7 TpH 5.5 260 248 270 TpH 5391 354 412 TpH 4.8 479 417 506 D 0 CFU/ml 1.02.10⁸ 2.79.10⁸ 1.47.10⁸ Ta= latency time (in minutes) Vmax = maximum rate (in pH units/minute) pHm= pH at the maximum acidification rate Tmax = time at Vmax (in minutes)pH 0 = pH at start of fermentation TpH 5.5 = time to arrive at pH 5.5(in minutes) TpH 5 = time to arrive at pH 5 (in minutes) TpH 4.8 = timeto arrive at pH 4.8 (in minutes) D 0 CFU/ml = quantity of bifidobacteriaobtained at the end of the fermentation.

It is noted that the acidification curve in the presence of cysteinediffers from the acidification curve in the presence of vitamin C, whichis itself virtually indistinguishable from the control acidificationcurve without vitamin C or cysteine. Given that vitamin C is anantioxidant, it is deduced from this that the stimulant effect ofcysteine is not an antioxidant effect but is more certainly an effect ofproviding essential amino acid.

Example 2 Determination of the Cysteine Stimulant Dose

A ferment comprising Streptococcus thermophilus (CNCM:I-2272)+Streptococcus thermophilus (CNCM: I-2773)+Streptococcusthermophilus (CNCM: I-2130)+Lactobacillus delbrueckii ssp. bulgaricus(CNCM: I-1519)-+Bifidobacterium animalis ssp lactis (CNCM: I-2494) isused.

“Milk models” are constituted by standard stirred yogurts comprising theferment described above.

The use of 0.2 μm-filtered cysteine was assessed in the “milk models” inproportions comprised from 5 mg/l to 50 mg/l (preferably from 5 to 20mg/l).

For the bifidobacteria counting method, reference may be made to M.Grand et al., Quantitative analysis and molecular identification ofbifidobacteria strains in probiotic milk products, Eur. Food Res.Technol. 217:90-92 (2003).

The population of bifidobacteria for the test containing the highestconcentration of L cysteine is 3.10⁸ CFU/ml at D+24 h, D correspondingto the time of packaging the product and remains stable afterpreservation for up to 28 days at 10° C. The standard control population(Control population D0: 1.10⁸ CFU/ml) is 9.10⁷ CFU/ml at 28 days ofpreservation at 10° C.

The population of bifidobacteria for the test containing the lowestconcentration of L cysteine is 1.10⁸ CFU/ml at D+24 h.

Certain products have an undesirable taste characterized by a sulphurnote which can be detected as from 0.002% of added cysteine. Below thiscysteine concentration the products are accepted: a dose of 0.0015%represents a good compromise between the organoleptic constraints andconstraints in terms of a Bifidobacterium population >2.10⁸ CFU/ml.

Growth tests on milk carried out in the presence of 0.0015% i.e. 15 mg/lof filtered cysteine have made it possible to reach a BifidobacteriumI-2494 population of 2.8.10⁸ CFU/ml after 28 days of preservation at 10°C. (the development of the population relative to the control withoutcysteine is represented in FIG. 2).

From a sensory analysis point of view, the products produced do not havea detectable undesirable taste in comparison with the standard.

Example 3 Acidification Kinetics

The ferment described in Example 2 is used.

The acidification kinetics of milk in the presence (15 mg/L) of theoptimum dose of cysteine and in the absence of cysteine (control) showthe absence of the effect of the cysteine on the overall kinetics.

Example 4 Effect of the Type of Treatment of the Sulphur-ContainingAmino Acid

The impact of sterilization by filtration or thermization of thecysteine and methionine is assessed (final concentration used: 50 mg/l).

The solutions are either filtered at 0.2 μm or autoclaved for 5 minutesat 121° C. then frozen in the form of beads in liquid nitrogen.

Constitution of the model:

Milex skimmed milk powder supplied by Arla food: 120 g

Water: quantity sufficient for 1 kg

Heat treatment: pasteurization for 30 minutes at 95° C. in a bubblingwater bath

Cysteine: supplier Sigma. The solution is prepared at 500 g/l, filteredon a 0.2 μm Nalgène filter unit or sterilized at 121° C. for 5 minutesby an autoclave controlled by temperature probe (Fetinge France S.A.,reference KL 60/101). This solution is injected sterilely into thepasteurized model for a final concentration of 50 mg/l.

Methionine: supplier Sigma. The solution is prepared at 300 g/l; thefiltration or sterilization treatment is identical to that carried outfor the cysteine solution. This solution is injected sterilely into thepasteurized model for a final concentration of 50 mg/l.

The seeding doses are referred to in Table 3 below.

TABLE 3 Seeding doses Volume for 1 l in μl Con- Filtered AutoclavedFiltered Autoclaved trol cysteine cysteine methionine methionine I-1630100 100 100 100 100 I-1519 + 220 220 220 220 220 I-1632 I-2494 95 95 9595 95 Filtered 100 cysteine Autoclaved 100 cysteine Filtered 100methionine Autoclaved 100 methionine

The seeding is 5.10⁶ CFU/ml of Streptococcus thermophilus and 5.10⁶CFU/ml of Lactobacillus bulgaricus.

The monitoring of the population of bifidobacteria in the modelpreserved at 4° C. as a function of the various conditions above isrepresented in FIG. 3 as well as in Table 4 below:

TABLE 4 Development of the population of bifidobacteria D 0 D 10 D 24 D29 CFU/ml CFU/ml CFU/ml CFU/ml Filtered 5.1.10⁸ 4.3.10⁸ 4.0.10⁸ 2.3.10⁸cysteine Autoclaved 3.1.10⁸ 5.2.10⁸ 2.8.10⁸ 1.7.10⁸ cysteine Filtered4.0.10⁸ 6.2.10⁸ 3.4.10⁸ 2.3.10⁸ methionine Autoclaved 3.4.10⁸ 3.5.10⁸3.5.10⁸ 2.8.10⁸ methionine Control 1.7.10⁸   7.10⁷   5.10⁷   2.10⁷

The reference time D0 corresponds to placing in pots (packaging). Themeasurements at D10, D24, D29 are carried out 10 days, 24 days, 29 daysrespectively after this placing in pots.

In all cases the population of bifidobacteria is increased by the supplyof cysteine or methionine. No effect of the heat treatment on theeffectiveness of the stimulants can be observed under the testconditions. The heat treatment applied to the cysteine at 50 mg/ldegrades only a part thereof, the residual concentration (not assessed)is sufficient to improve the population of bifidobacteria.

Example 5 Development of the Population of Bifidobacteria DuringPreservation in the Case of Incorporation of Cysteine BeforePasteurization

The dose of 12 mg/l of cysteine is defined as having a stimulant effectresponding to the target population (2.10⁸ CFU/ml) and respondingpositively in organoleptic terms (no detectable difference). Thisconcentration was assessed on direct incorporation in the model andpasteurized composition (95° C., 30 minutes).

The monitoring of the population of bifidobacteria in the product modelduring preservation at 10° C. is represented in FIG. 4.

The population of Bifidobacterium is 2.4.10⁸ CFU/ml at D1 (i.e. 24 hoursof storage) and remains stable after 44 days of preservation at 10° C.(above 1.4.10⁸ CFU/ml). The stimulant effect is clearly demonstratedrelative to the standard control (1.6.10⁸ CFU/ml at D1; 7.65.10⁷ CFU/mlat D8; 2.10⁷ CFU/ml at D28; 1.8.10⁷ CFU/ml at D35; 8.5.10⁶ CFU/ml atD44) under these conditions: the population at D0 is higher when thesulphur-containing amino acids are used and the maintenance of thepopulation during the life of the product is very much improved. Thisstimulant effect however remains less effective than the addition of 0.2μm filtered cysteine to the model (3.10⁸ CFU/ml), the heat treatmentresulting in a degradation of the cysteine (residual concentration lessthan 15 mg/l). An initial overdosage of the quantity of cysteine is tobe provided in the case where the cysteine undergoes a heat treatment.

Conclusions relating to the conditions of utilization of cysteine:

-   -   the utilization of directly filtered cysteine (with the ferment)        preserves the cysteine;    -   its addition to the composition of the heat-treated model        produces a slightly less good result in terms of population but        account must be taken of the degradation of the cysteine during        the heat treatment (less available);    -   its addition via a heat-treated dairy ingredient (for example        GlycoMacroPeptide corresponding to the fragment 106-169 of kappa        caseine) produces less good results (less available);    -   its addition in the frozen form to the ferment is possible.

Example 6 Production of a Fatty Stirred Yogurt According to theInvention on the Laboratory Scale (Micro-Production)

1. Composition of the Milk and Rehydration

The stirred yogurt comprises the following ingredients: skimmed milkwith 0% fat, cream with 40% fat and skimmed milk powder with 33%proteins.

Firstly, all the ingredients are combined together in order tostandardize the milk at a protein level (PL) of 4.4%, a fat level of3.5% (FL) and a dried matter level of 15.8% with stirring of the mediumfor 60 minutes at approximately 750 rpm with a HEIDOLPH® stirrer inorder for the proteins to rehydrate.

Control of the standardization is carried out with a MILKOSCAN FT 120®infrared detector from FOSS®. Below, an example of the necessaryquantities of each ingredient in order to obtain the targetscharacterizing the milk.

Ingredients In % Skimmed milk 0% fat 87.5 Cream with 40% fat 8.7 Skimmedmilk proteins 33% PL 3.8 TOTAL 100

2. Homogenization

The milk is then heated between 50° C. and 60° C. in order to melt thefat globules. Once the temperature is reached, the 10 litres arehomogenized with a MICROFLUIDIZER® from MICROCORPS®. This makes itpossible to break up the fat globules by passing the capillary milkthrough a grid under a pressure of 350 bars.

3. Pasteurization

A MEMMERT® water bath is prepared and adjusted to 103° C. The milk istransferred into 8 1-litre bottles, with a precise weighing of thisquantity for each bottle.

The bottles are immersed in the water bath up to the bottom of the neckat 103° C. for 35 minutes, then 10 minutes at 95° C. in the same waterbath.

4. Cooling and Storage

The bottles are cooled down in a cold water bath with a continuous flow,then stored at 4° C. in a refrigerator for 12 to 24 hours according tothe test schedule envisaged.

5. Holding

The milk bottles are removed from the refrigerator 45 minutes before theinoculation of the ferments and placed in a water bath at the consideredfermentation temperature, i.e. 37° C.

6. Fermentation

After inoculation of the ferments (5.10⁶ CFU/ml of Streptococcusthermophilus; 5.10⁶ CFU/ml of Lactobacillus bulgaricus; 5.10⁶ CFU/ml ofbifidobacteria) and L-cysteine (15 mg/l) at the fermentation temperatureof 37° C., the bottles are re-immersed in the water bath, and theacidification is monitored by a CINAC® from YSEBAERT® up to a pH of 4.8.

7. Cutting of Coagulum and Smoothing

The coagulum in the bottle is cut by hand. The yogurt with cut coagulumis poured into the hopper of the smoothing platform. The smoothing takesplace via a metal grid with a porosity of 500 microns and the smoothedproduct is cooled down to 20° C. via an exchange circuit in iced water.

8. Packaging and Storage

Packaging is carried out manually in 125-ml pots and the lid isheat-sealed with a DNV-100-25 PPV-A® heat sealer from FESTO®. Theproducts are stored in a cooler at 10° C. throughout the test.

Example 7 Assessment of the Dose of Cysteine to be Added in Order toObtain a Product with Good Organoleptic Quality and Containing theTarget Population of Bifidobacteria

Different products were prepared with increasing doses of cysteine (seethe table below).

The control was the standard dairy product containing the ferment.

Range: volume/1 L Cysteine dose 3.2 mL 0.0080% 80 mg/L 2 mL 0.0050% 50mg/L 0.8 mL 0.0020% 20 mg/L 0.4 mL 0.0010% 10 mg/L 0.2 mL 0.0005%  5mg/L

Each product was tasted by 4 individuals who were very familiar with thereference product from an organoleptic point of view. These individualsgave their opinions in terms of the presence of bad tastes (sulphurtaste, acid note), the reference being the standard product containingno cysteine.

Results

Test Populations (CFU/mL) Sensory assessment Time T0 T pH 4.8 Tf 495 min(n = 4) 1 4.40E+06 / 1.70E+08 Sulphur taste and/or acid note detected byall the tasters 0.008% 2 4.70E+06 1.90E+08 2.40E+08 Sulphur taste and/oracid note detected by all the tasters 0.005% 3 3.30E+06 2.50E+083.10E+08 Sulphur taste and/or acid note detected by all the tasters0.002% 4 / / / Sulphur taste and/or acid note detected by all thetasters 0.0015%  5 3.10E+06 1.50E+08 2.40E+08 No unpleasant tastedetected 0.001% 6 3.90E+06 1.10E+08 1.20E+08 No unpleasant tastedetected 0.0005% 

The 0.0015% dose not yet being optimum from an organoleptic point ofview, the 0.00125% dose was tested. This dose represents a very goodcompromise between the constraint in terms of population maintenance andthe constraint in terms of organoleptic quality.

The sensory profile of a product with 0.00125% (12.5 mg/l) addedcysteine was produced by a jury of experts comprising 15 individualstrained in this type of tasting.

Two repetitions were carried out. The tasters had to judge the productson the basis of 23 descriptors. The results based on these descriptors(essential for defining the organoleptic quality of the product relativeto the reference product) showed no significant harmful difference onthe basis of these descriptors. These descriptors were the following:

Product appearance

-   -   Visual whey (visual assessment of the quantity of whey on the        product surface)

Texture on the spoon before stirring

-   -   Shape holding ability (relates to the stability of the structure        of the product)

Texture on the spoon after stirring of the product

-   -   Thickness (resistance to the movement of the spoon)    -   Thread (continuity of the flow thread)    -   Covering (quantity of product which covers the back of the        spoon)

Texture in the mouth after stirring of the product

-   -   Melting away (speed of disappearance of the product in the        mouth)    -   Coating (coats the inside of the mouth)    -   Fat (Sensation of fat in the mouth)    -   Soft (Tactile sensation of softness in the mouth)

Flavours

-   -   Acid    -   Sweet    -   Bitter    -   Astringent

Milk flavourings

-   -   Unpleasant tastes    -   Cream    -   Butter    -   Milk    -   Fromage frais    -   Acetaldehyde    -   Lactoserum    -   Lactone    -   Lemon    -   Potato

The sought result is an absence of significant difference between thecontrol product and the product supplemented with cysteine.

In the present case, a product according to the invention, supplementedwith 12.5 mg/l of cysteine exhibits no significant difference in termsof appearance, texture, flavours and tastes relative to the controlproduct.

1-40. (canceled)
 41. Method for the preparation of a fermented foodproduct using ferments containing bifidobacteria, which fermented foodproduct has acceptable organoleptic properties, contains more than5×10⁷, bifidobacteria per gram of fermented food product for apreservation period of at least 30 days, and does not contain more than0.5% yeast extract or yeast autolysate using at least onesulphur-containing amino acid, at a total concentration of 5 to 75 mg/l,in the free form.
 42. Method according to claim 41 for the preparationof a fermented food product using ferments containing bifidobacteria,which fermented food product has acceptable organoleptic properties,contains more than 10⁸ bifidobacteria per gram of fermented food productfor a preservation period of at least 30 days and does not contain morethan 0.5% yeast extract or yeast autolysate using at least onesulphur-containing amino acid, at a total concentration of 5 to 75 mg/lin the free form.
 43. The method according to claim 41, for thepreparation of a fermented food product using ferments containingbifidobacteria, which fermented food product does not contain more than0.5% yeast extract or yeast autolysate using at least onesulphur-containing amino acid, at a total concentration of 5 to 20 mg/lin the free form.
 44. Method for the preparation of a fermented foodproduct using ferments containing bifidobacteria, which fermented foodproduct has acceptable organoleptic properties, contains more than5×10⁷, bifidobacteria per gram of fermented food product for apreservation period of at least 30 days, and does not contain more than0.5% yeast extract or yeast autolysate using at least onesulphur-containing amino acid, at a total concentration of 5 to 30 mg/l,in the free form.
 45. The method according to claim 44, for thepreparation of a fermented food product using ferments containingbifidobacteria, which fermented food product has acceptable organolepticproperties, contains more than 10⁸ bifidobacteria per gram of fermentedfood product for a preservation period of at least 30 days, and does notcontain more than 0.5% yeast extract or yeast autolysate using at leastone sulphur-containing amino acid, at a total concentration of 5 to 30mg/l in the free form.
 46. The method according to claim 44, for thepreparation of a fermented food product using ferments containingbifidobacteria, which fermented food product does not contain more than0.5% yeast extract or yeast autolysate using at least onesulphur-containing amino acid, at a total concentration of 12 to 15 mg/lin the free form.
 47. Fermented food product, having acceptableorganoleptic properties, containing ferments comprising more than 5×10⁷,bifidobacteria per gram of fermented food product for a preservationperiod of at least 30 days, and having a total concentration ofsulphur-containing amino acids in the free form of 5 to 50 mg/l.
 48. Thefermented food product according to claim 47, having acceptableorganoleptic properties, containing ferments comprising more than 10⁸bifidobacteria per gram of fermented food product for a preservationperiod of at least 30 days, and having a total concentration ofsulphur-containing amino acids in the free form of 5 to 50 mg/l.
 49. Thefermented food product according to claim 47, having acceptableorganoleptic properties, and having a total concentration ofsulphur-containing amino acids in the free form of 5 to 30 mg/l.
 50. Thefermented food product according to claim 47, having acceptableorganoleptic properties, and having a total concentration ofsulphur-containing amino acids in the free form of 5 to 20 mg/l.
 51. Thefermented food product according to claim 47, having acceptableorganoleptic properties, and having a total concentration ofsulphur-containing amino acids in the free form of 10 to 15 mg/l. 52.The fermented food product according to claim 47, having acceptableorganoleptic properties, and having a total concentration ofsulphur-containing amino acids in the free form of 12 to 15 mg/l. 53.The fermented food product according to claim 47, having acceptableorganoleptic properties, and having a total concentration ofsulphur-containing amino acids in the free form of 12.5 mg/l.
 54. Thefermented food product according to claim 47, in which the ratio of thenumber of bifidobacteria contained in the fermented food product at theend of the preservation period to the number of bifidobacteria containedin the fermented food product at the start of the preservation period ofat least 30 days, is 0.2 to 0.8.
 55. Fermented food product preservedfor a preservation period of at least 30 days, at a temperature of 4 to10° C., having acceptable organoleptic properties and containingferments comprising more than 5×10⁷, bifidobacteria per gram offermented food product.
 56. The fermented food product according toclaim 55, containing ferments comprising more than 10⁸ bifidobacteriaper gram of fermented food product.
 57. The fermented food productaccording to claim 47, containing 5 to 50 mg/l, of sulphur-containingamino acids and 5 to 50 mg/l, of cysteine and/or 5 to 30 mg/l, ofmethionine.
 58. The fermented food product according to claim 47,containing less than 0.5% (w/w) of substances containing more than 1.7%sulphur-containing free amino acids.
 59. The fermented food productaccording to claim 47, containing less than 0.5% (w/w) of yeast extractand/or yeast autolysate and/or milk, plant or soya protein hydrolysate.60. The fermented food product according to claim 47, in which thebifidobacteria are of the type Bifidobacterium animalis.
 61. Thefermented food product according to claim 60, in which theBifidobacterium animalis, is selected from the group consisting ofBifidobacterium animalis animalis and/or Bidifobacterium animalislactis, and/or Bifidobacterium breve and/or Bifidobacterium longumand/or Bifidobacterium infantis and/or Bifidobacterium bifidum.
 62. Thefermented food product according to claim 47, based on plant juiceselected from the group consisting of fruit juice or vegetable juicesuch as soya juice, or on a dairy product, selected from the groupconsisting of cow's milk and/or goat's milk.
 63. The fermented foodproduct according to claim 47, in which the ferments contain lacticbacteria.
 64. The fermented food product according to claim 63, in whichthe ferments contain one or more bacteria of the genus Lactobacillusspp. selected from the group consisting of Lactobacillus delbrueckiibulgaricus and/or Lactobacillus casei and/or Lactobacillus reuteriand/or Lactobacillus acidophilus and/or Lactobacillus helveticus and/orLactobacillus plantarum, and/or bacteria of the type Lactococcuscremoris and/or Streptococcus thermophilus and/or Lactococcus lactisand/or one or more bacteria of the genus Leuconostoc.
 65. The fermentedfood product according to claim 47, in which the proportion ofbifidobacteria in the ferments is 20 to 80%.
 66. The fermented productaccording to claim 47, presented in the form of a stirred fermented foodproduct or a fermented food product for drinking or an infant fermentedfood product.
 67. Process for the preparation of a fermented foodproduct from a starting substance, comprising a stage of seeding astarting substance, optionally pasteurized, by inoculation with seedingferments containing bifidobacteria, in order to obtain a seededsubstance, a stage of fermentation of the seeded substance obtained inthe preceding stage in order to obtain a fermented substance, a stage ofincorporation of at least one sulphur-containing amino acid in the freeform at a concentration of 5 to 75 mg/l, this stage of incorporationbeing able to occur either before the seeding stage, or substantiallysimultaneously with the seeding stage, or after the seeding stage andbefore the fermentation stage, providing that the fermented food productdoes not contain more than 0.5% (w/w) of yeast extract and/or yeastautolysate.
 68. The process according to claim 67, not comprising astage of addition of additional substances containing one or moresulphur-containing amino acids.
 69. The process according to claim 67,comprising a stage of addition of additional substances containing oneor more sulphur-containing amino acids in the free form, theconcentration of sulphur-containing amino acids in the free form in theadditional substances being less than 1.7%, preferably less than 0.5%,and the concentration of said additional substances in the fermentedfood product being less than 0.5%.
 70. The process according to claim67, comprising a stage of addition of additional substances constitutedby a yeast extract and/or yeast autolysate and/or milk, plant or soyaprotein hydrolysate at a concentration of less than 0.5% (w/w).
 71. Theprocess for the preparation of a fermented food product according toclaim 67, also comprising a pasteurization stage taking place before theseeding stage, making it possible to obtain a pasteurized startingsubstance from the starting substance.
 72. The process for thepreparation of a fermented food product according to claim 71, in whichthe stage of incorporation of at least one sulphur-containing amino acidtakes place before the pasteurization stage, the sulphur-containingamino acid or acids being incorporated at a concentration of 5 to 75mg/l.
 73. The process for the preparation of a fermented food productaccording to claim 67, in which the stage of incorporation of at leastone sulphur-containing amino acid takes place substantiallysimultaneously with the seeding stage, the sulphur-containing amino acidor acids being incorporated at a concentration of 5 to 50 mg/l.
 74. Theprocess for the preparation of a fermented food product according toclaim 67, in which the stage of incorporation of at least onesulphur-containing amino acid takes place after the seeding stage andbefore the fermentation stage, the sulphur-containing amino acid oracids being incorporated at a concentration of approximately 5 toapproximately 50 mg/l.
 75. The process for the preparation of afermented food product according to claim 67, comprising a stage ofaddition of an intermediate preparation simultaneously with the seedingstage or between the seeding stage and the fermentation stage, so as toobtain, from the seeded substance, a completed seeded substance, orafter the fermentation stage, so as to obtain, from the fermentedsubstance, a completed fermented substance, said intermediatepreparation comprising a preparation of fruits and/or cereals and/oradditives selected from the group consisting of flavourings andcolourings, and said stage of addition of an intermediate preparationcan take place simultaneously with the stage of incorporation of atleast one sulphur-containing amino acid.
 76. The process for thepreparation of a fermented food product according to claim 67, in whichthe seeding stage comprises inoculation with seeding ferments containing10⁶ to 2×10⁸, bifidobacteria, per ml of starting substance.
 77. Theprocess for the preparation of a fermented food product according toclaim 67, in which the bifidobacteria are chosen from bacteria of thetype Bifidobacterium.
 78. The process for the preparation of a fermentedfood product according to claim 77, in which the bacteria of the typeBifidobacterium animalis are selected from the group consisting ofBifidobacterium animalis animalis and/or Bidifobacterium animalislactis, and/or Bifidobacterium breve and/or Bifidobacterium longumand/or Bifidobacterium infantis and/or Bifidobacterium bifidum.
 79. Theprocess for the preparation of a fermented food product according toclaim 67, in which the seeding ferments contain lactic bacteria.
 80. Theprocess for the preparation of a fermented food product according toclaim 79, in which the seeding ferments contain one or more bacteria ofthe genus Lactobacillus spp. selected from the group consisting ofLactobacillus delbrueckii bulgaricus and/or Lactobacillus casei and/orLactobacillus reuteri and/or Lactobacillus acidophilus and/orLactobacillus helveticus and/or Lactobacillus plantarum, and/or bacteriaof the type Lactococcus cremoris and/or Streptococcus thermophilusand/or Lactococcus lactis and or one or more bacteria of the genusLeuconostoc.
 81. The process for the preparation of a fermented foodproduct according to claim 67, in which the proportion of bifidobacteriain the seeding ferments is 20 to 75%.
 82. The process for thepreparation of a fermented food product according to claim 67, in whichthe starting substance is based on plant juice selected from the groupconsisting of fruit juice or vegetable juice such as soya juice, or on adairy product, and selected from the group consisting of cow's milkand/or goat's milk.
 83. The process for the preparation of a fermentedfood product according to claim 67, in which the starting substance is apasteurized starting substance, which is held, optionally homogenized,and cooled down, obtained from a raw material, said process comprising,before the seeding stage, the following successive stages: a stage ofstandardization of fatty substances of the raw material so as to obtaina standardized substance, a stage of enrichment with dried matter of thestandardized substance obtained in the preceding stage, so as to obtainan enriched substance, a stage of pre-heating of the enriched substanceobtained in the preceding stage, so as to obtain a starting substance, astage of pasteurization and holding of the starting substance obtainedin the preceding stage, so as to obtain a pasteurized and heldsubstance, an optional stage of homogenization of the pasteurized andheld substance obtained in the preceding stage, so as to obtain apasteurized, held and optionally homogenized substance, a stage ofinitial cooling of the pasteurized, held and optionally homogenizedsubstance obtained in the preceding stage, so as to obtain a pasteurizedstarting substance, held, optionally homogenized, and cooled.
 84. Theprocess for the preparation of a fermented food product according toclaim 67, comprising a packaging stage between the seeding stage and thefermentation stage, said packaging stage making it possible to obtain,from the seeded substance obtained in the seeding stage, a seeded andpackaged substance.
 85. The process for the preparation of a fermentedfood product according to claim 67, comprising: a stage of seeding astarting substance, optionally pasteurized, by inoculation with seedingferments containing 10⁶ to 2×10⁸ bifidobacteria, per ml of startingsubstance, in order to obtain a seeded substance, a stage of packagingthe seeded substance obtained in the preceding stage, in order to obtaina packaged seeded substance, a stage of fermentation of the packagedseeded substance obtained in the preceding stage, such that thetemperature at the start of fermentation is 36 to 43° C., thetemperature at the end of fermentation is 37 to 44° C., and thefermentation time is 6 to 11 hours, in order to obtain a fermentedsubstance, a stage of final cooling of the fermented substance obtainedin the preceding stage, such that the temperature at the start of thefinal cooling is less than 22° C. and the temperature at the end of thefinal cooling is 410° C., so as to obtain a fermented food product. 86.The process for the preparation of a fermented food product according toclaim 85, in which the a stage of seeding a starting substance,optionally pasteurized, by inoculation with seeding ferments contains10⁶ to 10⁷ bifidobacteria per ml of starting substance, in order toobtain a seeded substance.
 87. The process for the preparation of afermented food product according to claim 67, comprising the followingsuccessive stages after the fermentation stage: a stage of intermediatecooling of the fermented substance obtained in the fermentation stage,so as to obtain a pre-cooled substance, a stage of storage of thepre-cooled substance obtained in the preceding stage, so as to obtain astored substance, a stage of final cooling of the stored substanceobtained in the preceding stage, so as to obtain a fermented foodproduct.
 88. The process for the preparation of a fermented food productaccording to claim 87, in which the fermentation stage is such that thetemperature at the start of fermentation is 36 to 43° C., and thefermentation time is 6 to 11 hours.
 89. The process for the preparationof a fermented food product according to claim 87 in which theintermediate cooling stage is such that the intermediate cooling time is1 hour to 4 hours and the intermediate cooling temperature is 4 to 22°C.
 90. The process for the preparation of a fermented food productaccording to claim 87 in which the storage stage is such that thestorage time is less than or equal to 40 hours.
 91. The process for thepreparation of a fermented food product according to claim 87, in whichthe final cooling stage is such that the temperature at the start offinal cooling is less than 22° C. and the temperature at the end offinal cooling is 4 to 10° C.
 92. The process for the preparation of afermented food product according to claim 87, comprising: a stage ofseeding a starting substance, optionally pasteurized, by inoculationwith seeding ferments containing 10⁶ to 2.10⁸, bifidobacteria per ml ofstarting substance in order to obtain a seeded substance, a stage offermentation of the seeded substance obtained in the preceding stage,such that the temperature at the start of fermentation is 36 to 43° C.,the temperature at the end of fermentation is 37 to 44° C., and thefermentation time is 6 to 11 hours, in order to obtain a fermentedsubstance, a stage of intermediate cooling of the fermented substanceobtained in the preceding stage, such that the intermediate cooling timeis 1 hour to 4 hours, and the intermediate cooling temperature is 4 to22° C., so as to obtain a pre-cooled substance, a stage of storage ofthe pre-cooled substance obtained in the preceding stage, such that thestorage time is less than or equal to 40 hours, so as to obtain a storedsubstance, a stage of final cooling of the stored substance obtained inthe preceding stage, such that the temperature at the start of finalcooling is less than 22° C. and the temperature at the end of finalcooling is 4 to 10° C. so as to obtain a fermented food product.
 93. Theprocess for the preparation of a fermented food product according toclaim 91, in which the stage of seeding a starting substance, optionallypasteurized, by inoculation with seeding ferments contains 10⁶ to 10⁷bifidobacteria per ml of starting substance in order to obtain a seededsubstance.
 94. The process for the preparation of a fermented foodproduct according to claim 87, comprising an additional stirring stagebetween the fermentation stage and the intermediate cooling stage,making it possible to obtain, from the fermented substance obtained inthe fermentation stage, a stirred fermented substance.
 95. The processfor the preparation of a fermented food product according to claim 87,comprising after the final cooling stage, a stage of preservation of thefermented food product at a temperature comprised between 4 and 10° C.96. Fermented food product as obtained from the process of claim 67.